The present invention relates to methods for preserving biological tissue specimens to be subject to infrared spectroscopy for detecting a malignant or premalignant anomaly, and for removing the effects of polymorphs for infrared spectroscopic detection of cellular anomalies.
It has been well documented that infrared spectra of various human cancers and precancerous lesions differ substantially from those of the corresponding normal tissues and cells. Several attempts have been made to develop infrared spectroscopic methods for the screening of cytological anomalies, in particular the screening of cervical malignancy or premalignant anomalies. The commonly used cytological screening methods, such as the Pap smear test, are mainly based on the morphological changes of cells, whereas the infrared spectroscopic method is based on the structural changes at the molecular level in cells. Since structural changes at the molecular level occur before morphological changes in abnormal cells, the infrared spectroscopic method is expected to be more accurate than the conventional cytology method in terms of early detection of malignant and premalignant anomalies. A recent clinical study (Ref. 11) has shown that the false negative rate of the infrared spectroscopic method for the screening of the neoplastic cervical cells is about ten times better than the traditional Pap smear test. However, there are still two main obstacles in the infrared spectroscopic method, which cause an extremely high false positive rate and prevent the infrared spectroscopic method from being acceptable for routine cytological screening. These two obstacles relate to problems caused by polymorph effects and in sample preparation.
In many areas of the world, the majority of the cervical specimens exhibit positive results when the infrared spectroscopic method is used. After many years research, we found that this extremely high false positive rate was essentially due to the effects of polymorphs. When a cervical specimen exhibits mild to moderate inflammation, the changes in the molecular arrangement and structure in the cells are insignificant but a considerable amount of polymorph cells are present in the cervical specimen. The presence of a large amount of polymorphs in the cervical cell specimen not only prevents the search for abnormal cervical cells in the specimens under a microscope by the conventional Pap smear test, but also prevents the infrared spectroscopic method from distinguishing a normal cervical specimen from an abnormal one. When polymorphs are present in a cervical specimen, the resulting infrared spectrum is a superimposed spectrum composed of the infrared spectra of cervical cells and polymorph cells. The infrared spectrum of polymorph cells has many features similar to those in the infrared spectra of various precancerous and atypical cells. Consequently, in the presence of polymorphs, a non-neoplastic cervical cell specimen will show an infrared spectrum similar to that of the precancerous or atypia cervical cells, which will lead to a false positive diagnosis.
In many areas of the world, cervical specimens with mild to moderate inflammation are very common place due to the specific environmental effects and sexual behavior in these areas. Therefore, a majority of the cervical specimens from the general public in these areas are accompanied with a significant amount of polymorphs. Consequently, the infrared spectroscopic method may lead to a false positive diagnosis for a majority of the population. This false positive effect of polymorphs must be removed before the infrared spectroscopic method can be adopted to the screening of cervical anomalies for the general population.
Another obstacle in the infrared spectroscopic method is problems in the sample preparation. At the present time, there are several methods to prepare cellular samples for infrared spectroscopic study.
The most common method is the wet process. In this process, exfoliated fresh cells are suspended in saline, centrifuged into a cellular pellet, and some of the wet pellet is then placed on an infrared spectroscopic sample holder for the infrared spectroscopic measurement and analysis.
Benedetti et al., G. Appli. Spectrosc., 44: 1276-1280 (1990) have adopted a method of infrared spectroscopic study for powdered solid samples in which they prepared dry solid powder of fresh biological cells for infrared spectroscopic analysis. In this dry process, fresh lymphocytes were separated from other constituents in the blood by chemicals, and the fresh lymphocytes were dried into a solid. The solid lymphocytes were ground into fine powder with KBr powder. The mixture of solid lymphocyte and KBr was then pressed into a clear solid pellet for infrared spectroscopic analysis. The infrared spectroscopic results obtained by this process are usually inaccurate due to the fact that grinding destroys the cellular form of lymphocytes, creating structural changes at the molecular level which mask those arising from neoplasm and other diseases, and decompose some biomolecules in the cells by the heat generated from the grinding.
In the dry process described by Gal et al., Anticancer Research, 14: 1541-1548 (1994), fresh cultured cells were washed and suspended in normal saline, smeared on a ZnSe window, evaporated for 10 minutes at 37.degree. C. and then the infrared spectrum of the exposed cellular proteins was measured. It is evident from Example 1 that in this process, the intermolecular structure in the cells was destroyed by the hypertonic crenation during the heating and drying process and thus no infrared spectrum of most of the important cellular molecules could be obtained. Only the infrared spectra of the exposed cellular proteins could be measured.
All the present wet and dry sample preparation methods for infrared spectroscopic study of tissue cells are dealing with fresh cells without any preservation. If the fresh cell specimens are not used immediately for infrared spectroscopic analysis, for instance cellular specimens are transported to the pathology laboratory before they are prepared for infrared spectroscopic analysis, the fresh cell specimens must be kept frozen until they are ready for analysis. At room temperature, cellular specimens either in the fresh form or in saline solution will deteriorate very fast. The infrared spectra of deteriorating cells have features similar to those in the spectra of abnormal cells and will lead to a false diagnosis and an increase in the false positive rate.
In many countries, screening of cervical anomalies is done in central laboratories. The transportation of the cervical specimens from the clinics to the central laboratory in the frozen form is extremely impractical. Moreover, in common practices, the cervical cell specimens after preparation are required to be kept for several years for future references in hospitals and clinics. One way to keep the cervical specimens for several years is to store the specimens in a liquid nitrogen tank. For a large number of specimens, such as cervical specimens for screening, to keep the specimens in liquid nitrogen is extremely impractical. The best way to resolve these problems is to fix cellular specimens by preservatives.
The criteria for the selection of preservatives and methods of preservative treatment, which are suitable for the detection of anomalies in tissue cells by infrared spectroscopic technology, are as follows: (1) The preservative must not have any chemical reaction with biomolecules in tissue cells to cause structural changes at the molecular level; (2) The preservative treatment must not damage the intermolecular arrangement and intramolecular structure in cells, which are the basis of detection of anomalies in cells by the infrared spectroscopic method; and (3) The preservative must not have infrared absorption bands at the same frequency regions as the infrared absorption bands of biological tissue cells. Otherwise, the infrared spectra of tissue cells are masked by the infrared spectra of the preservatives, which makes the analysis of the infrared spectra of cells to detect cellular anomalies impossible. Unfortunately, all the conventional preservatives (or fixatives) for biological tissues and cells in pathology and cytology, such as alcohol, formalin, nitrates, etc. do not meet these criteria.
In Hook et al., J. Microscopy, 141: 69-78 (1985) cell samples were prepared for the determination of the amount of magnesium ion in cells. The cells were suspended in an ammonium nitrate solution or buffered saline glucose solution, deposited on the analysis support and then air dried. This sample preparation method can not be adopted for the infrared spectroscopic method because both ammonium nitrate and glucose strongly interact with cellular molecules and cause molecular rearrangement and structural changes in tissue cells. Moreover, both ammonium nitrate and glucose have infrared absorption bands in the frequency regions of the infrared absorption bands of tissue cells and thus interfere with the spectral analysis of cells. Hook et al. were only interested in the determination of the amount of magnesium ion in cells. Therefore, either the damage of the cellular structural or interactions of cellular molecules with preservatives and other molecules in the sample preparation were not their concern.
In Sinor et al., U.S. Pat. No. 5,030,560, issued Jul. 9, 1991, cells were preserved with a drying solution consisting of monosaccharide, disaccharide, trisaccharide or cyclitol and a salt. In their sample preparation method, the washed cells must be deposited onto a dye coated solid-phase support in the form of a monolayer of cells. Then, the drying solution was added to the surface of the solid-phase support containing the cell monolayer. After a period of incubation, the excess drying solution was removed from the cell monolayer. Finally, the remaining drying solution was absorbed by the dessicant material in a sealed container for 3-8 days at 2-8.degree. C. Cells were lysed before the application of the drying solution, which allowed the drying solution better access to the inside of the cells. Saccharides and cyclitol in their drying solution strongly interact with cellular molecules and change the structure and arrangement of cellular molecules. Saccharides and cyclitol also have strong absorption bands in the infrared frequency regions of the absorption bands of biological tissues and cells. The lysis procedure in this method also changes the molecular structure and arrangement in cells. Therefore, this drying solution is not suitable for the infrared spectroscopic study of biological cells. The interaction of saccharides with cellular molecules and the changes of intermolecular structures in the cells were not the concerns of Sinor and Eatz because their cell preparation was for immunoassays which takes place on the surface of cells. Moreover,the drying process in the Sinor et al. procedure is too tedious and not practical for a routine screening of cervical specimens by the infrared spectroscopic method.
It is an object of the present invention to provide a simple and effective method for the infrared spectral preservation of tissue cells for infrared spectroscopic analysis.
It is a further object to spectrally preserve the tissue cells such that they can be safely stored for years at room temperatures.
It is a still further object of the invention to provide a process to remove the polymorph effects from the infrared spectra of negative specimens in order to reduce the false positive rate to such a level that the use of the infrared spectroscopic method for the screening of cellular anomalies becomes practically possible.